L-n-acyl-o-ethylthreonine

ABSTRACT

THIS DISCLOSURE RELATES TO NEW COMPOUNDS REREPRESENTED BY O-ETHYL THREONINE, AS WELL AS N-SUBSTITUTED AND CARBOXY-SUBSTITUTED DERIVATIVES THEREOF. SEVERAL OF THE COMPOUNDS, AND ESPECIALLY THE L-ISOMERS, ARE USEFUL IN THE TREATMENT OF LIVESTOCK TO CONTROL INFECTION BY PLEUROPNEUMONIA-LIKE ORGANISMS.

Int. Cl. C07c 101/04 US. Cl. 260501.11 1 Claim ABSTRACT OF THEDISCLOSURE This disclosure relates to new compounds represented byO-ethyl threonine, as well as N-substituted and carboxy-substitutedderivatives thereof. Several of the compounds, and especially theL-isomers, are useful in the treatment of livestock to control infectionby pleuropneumonia-like organisms.

BACKGROUND OF THE INVENTION Field of the invention This inventionrelates to novel derivatives of threonine and to the use of the same incombating pleuropneumonialike organisms in livestock.

Description of the prior art This application is a division ofapplicants copending US. application of Ser. No. 692,699, filed Dec. 22,1967, and issued as US. Pat. 3,639,382 on Feb. 1, 1972.

One of the most serious problems in raising livestock and particularlyfowl, swine and cattle, is infection by pleuropneumonia-like organismswhich are responsible for a number of chronic respiratory diseases. Inthe fowl industry alone, it is estimated that pleuropneumonia-likeorganisms are responsible for losses of the order of 100 million dollarsper year. Thus, there is a clear need for an agent which is capable ofpreventing and combating infections caused by said organisms.Additionally, pleuropneumonia-like organisms are indicated as a cause ofhuman disabilities such as urogenital tract infections and arthritis.

Heretofore, there has been no commercial product which is completelysatisfactory for routine prophylactic use against these organisms inlivestock. A few compositions have been used therapeutically, but nonehas been found fully satisfactory even for this limited utility.Accordingly, there has not previously been available in the art any modeof treatment which satisfactorily combats or prevents livestockinfections from pleuroneumoniatype organisms.

SUMMARY OF THE INVENTION The present invention provides a novel class ofthreonine derivatives which exhibit a marked and unexpected activityagainst pleuropneumonia-like organisms (PPLO) The invention alsoprovides methods for the prophylactic and therapeutic treatment oflivestock. The invention further provides novel intermediates useful inhe preparation of the PPLO-active compounds.

The compounds of this invention having anti-PPLO activity are L-O-ethylthreonine and derivatives thereof. In general, they can be representedby the formula NHRz CHsCHHC R3 wherein R is selected from the groupconsisting of hydrogen and substituents which are cleavable by bodyprocesses, e.g. aminoalkyl acyl and R is selected from the United StatesPatent 0 ICC group consisting of hydroxy, lower alkoxy, monocyclicaryloxy, preferably monocarbocyclic aryloxy, amino, hydroxamino andhydrazino. The principal active form of the O-ethyl threonine compoundsis believed to be that in which the amino nitrogen is unsubstituted. Thecompounds are, therefore, administered in either the free amino form orin a form in which the amino nitrogen bears a substituent which isreadily cleaved by body processes. The class of substituents found to bemost useful in this latter regard are the acyl residues of amino acids,i.e. aminoalkyl acyl moieties of compounds such as alanine, lysine,leucine, cysteine, proline, phenylalanine, glycine, isoleucine, proline,valine, etc. The most preferred compounds for animal treatment are thosein which the amino nitrogen is unsubstituted.

The substituent R can be hydroxy, lower alkoxy, monocyclic aryloxy,amino, hydroxamino or hydrazino and the resulting O-ethyl threoninecompound will be, respectively, the free acid, a lower alkyl ester, anaryl ester, an amide, an N-hydroxyamide or a hydrazide. As used herein alower alkyl group is an alkyl of 1-l0 carbon atoms, and preferably about1-6 carbon atoms. The lower alkoxy, monocyclic aryloxy, amino,hydroxamino and hydrazino groups include those which are inertlysubstituted, i.e. which bear substituents which do not adversely affectthe use of the compounds in the treatment of animals. Such substituentsmay include alkyl, aryl, halo, amino, hydroxy, cyclized alkylene, ester,nitro,

haloalkyl, alkoxy, etc. Thus, R can be, for example,

lower alkoxy such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,sec-butoxy, isobutoxy, amyloxy, hexoxy, cyclohexoxy, heptoxy, octoxy anddecoxy; amino lower alkoxy such as 2-aminoethoxy, 3-aminopropoxy,3-amino n butoxy, methylaminoethoxy and dimethylaminoethoxy; hydroxylower alkoxy such as Z-hydroxyethoxy, 3-hydroxypropoxy and3-hydroxy-n-butoxy; alkoxy lower alkoxy such as ethoxymethoxy,ethoxyethoxy, ethoxy-n-propoxy; aryl lower alkoxy such as benzyloxy and2-phenylethoxy; haloalkoxy such as 2-chloroethoxy and3-chloro-n-propoxy; cycloalkyl lower alkoxy such as cyclohexylmethoxy;monocyclic aryloxy such as phenoxy; alkyl monocyclic aryloxy such astoloxy and ethylphenoxy; amino monocyclic aryloxy such as aminophenoxy,ethylaminophenoxy and dimethylaminophenoxy; halo monocyclic aryloxy suchas chlorophenoxy and dichlorophenoxy; hydroxy monocyclic aryloxy such ashydroxyphenoxy; nitro monocyclic aryloxy such as nitrophenoxy; nitromonocyclic aryloxy such as nitrophenoxy; haloalkyl monocyclic aryloxysuch as chloromethyl phenoxy and trifiuoromethyl phenoxy; alkoxymonocyclic aryloxy such as anisoxy and ethoxyphenoxy; amino; alkylaminosuch as methylamino, ethylamino, n-propylamino, isopropyl-amino,n-butylamino, dimethylamino, benzylamino and diethylamino; arylaminosuch as anilino; hydroxamino; hydrazino; and cyclized alkylene aminosuch as N-piperidyl and N-morpholinyl.

Examples of compounds of the invention include the following:

O-ethyl threonine;

ariginyl-O-ethyl threonine;

leucyl-O-ethyl threonine;

lysyl-O-ethyl threonine;

glycyl-O-ethyl threonine;

methyl threo-2-amino-3-ethoxybutyrate;

ethyl threo-2-amino-3-ethoxybutyrate; isopropylthreo-2-amino-3-ethoxybutyrate; n-butyl threo-2-amino-3-ethoxybutyrate;methyl threo-2-arginylamino-3-ethoxybutyrate; ethylthreo-2-lysylamino-3-ethoxybutyrate; n-propylthreo-2-prolylamino-3-ethoxybutyrate; threo-2-amino-3-ethoxybutyramide;N-methyl-threo-Z-amino-3-ethoxybutyramide;

Patented Mar. 12, 1974 N-ethyl-threo-2-amino-3-ethoxybutyramide;

N-methyl-threo-lysylamino-3-ethoxybutyrarnide;N-(threo-2-amino-3-ethoxybutyryl) piperidine;N-(threo-2-amino-3-ethoxybutyryl) morpholine;N-hydroxy-threo-2-amino-3-ethoxybutyramide;N-hydroxy-thre-2-leucylamino-3-ethoxybutyramide;N-methoxy-threo-Z-amino-3-ethoxybutyramide;N-ethoxy-threo-Z-amino-3-ethoxybutyramide; threo-Z-amino 3-ethoxybutyricacid hydrazide; threo-Z-amino-3-ethoxybutyric acid methyl hydrazide;threo-2-amino-3-ethoxybutyric acid ethyl hydrazide;threo-2-valylamino-3-ethoxybutyric acid hydrazide;

etc. The presently preferred compounds are those wherein R is hydroxyl,i.e. the free carboxylic acids such as O-ethyl threonine. The compoundsmay also be used in the form of non-toxic salts, including thehydrochloride, sulfate, etc. salts of the amine group and the sodium,ammonium, calcium etc. salts of the carboxylic acid group.

It has been found that the products having anti-PPLO activity are theL-stereoisomers of the noted O-ethyl threonine compounds. The D-isomersexhibit little or no activity. It is possible to use either mixtures ofthe D and L forms, such as the substantially equimolar racemic mixture,or the L-isomer in the treatment of animals, but

the latter is preferred. It has also been found that the correspondingallothreonine derivatives are substantially devoid of anti-PPLOactivity. The O-n-propyl, O-isopropyl, O-n-butyl and higher alkylthreonine derivatives are inactive and the O-methyl derivatives exhibita very low order of activity such that their use as anti-PPLO agents isnot feasible. Accordingly, the high order of activity possessed by theinstant compounds is both unique and unexpected. In addition, thecompounds of this invention have been found effective in combattingcoccidiosis and malaria and gram-positive bacteria such as thosecommonly found in turkeys, infected with PPLO. It is a particularadvantage that the compounds are effective against resistant strains ofPPLO which are not susceptible to treatment by known agents.

The active compounds are readily prepared from available startingmaterials, particularly from crotonic acid and derivatives thereof. Forexample, a suitable route to DL-O-ethyl threonine is shown in thefollowing reaction sequences.

cm 1 Hg(OAc)z on. 002115 on. /OC2H5 on OH NHa on (U: canon 1 I H B 5 /CEr 00 OH 2 Br 0 0 or: NH; 0 0 OH In the process illustrated in reactionsequence I, crotonic acid, mercuric acetate and ethanol are heated untilall of the solids are dissolved and the solution is cooled to recoverthe 2-acetoxy mercuri-3-ethoxybutyric acid as a solid precipitate. Theprecipitate is brominated in water with bromine and potassium bromidewhile illuminated by a light source. The solution is then acidified andextracted to give 2-bromo-3-ethoxybutyric acid. The bromoacid compoundis converted to the corresponding free amine by reaction with ammoniumhydroxide to yield a mixture of three and erythro derivatives.Separation of the threo isomer is accomplished by reaction with formicacid to give the N-formyl derivative which is recovered by fractionalcrystallization. The N-formyl compound can then be cleaved with acid togive O-ethyl threonine, from which the various N- andcarboxy-substituted derivatives are prepared by standard techniques.

on. on, 00211 on. 00.11,

CH CzHrOH CH H2 C -r E N02 000R. NO; 090115 NH; ooorr Reaction sequenceII illustrates the preparation of O- ethyl threonine from an ester of2-nitrocrotonic acid by ethoxylation of the alpha-nitro double bond withethanol, to give the corresponding ester of 2-nitro-3-ethoxybutyric acidfollowed by controlled reduction of the nitro group, for example, byhydrogenation over platinum oxide. The group R is the residue of anorganic alcohol which can be readily cleaved by hydrolysis orhydrogenation; such as an aromatic or t-butyl group.

Separation of the three isomer and preparation of derivatives thereofcan be accomplished as above.

(III) CH3 H3 0 2115 CH CzHaOH CH I] CH Rr-NH COOR5 R4N COOR5 CH3 OCzHs c(1H NHr COOH Reaction sequence III illustrates the preparation ofO-ethyl threonine from an ester of an N-substituted 2- aminocrotonicacid in which R; is an acyl group and R is the residue of an alcohol,both being readily cleaved, as by hydrolysis, to give the free amino,free carboxylic acid compound. In this sequence, R will preferably be anaromatic group, e.g. phenyl, to facilitate the cleavage.

013 O CzH5 CH 0 02115 CH 0 CzHs CH NHs CH H1O C l CHO HCN /CE 1101 /CHNHz CN NH: C O OH Reaction sequence IV illustrates the preparation of O-ethyl threonine from 2-ethoxypropionaldehyde by reaction with ammoniaand hydrogen cyanide or ammonium chloride and alkali cyanide to form them-aminonitrile, followed by hydrolysis of the nitrile group to thecarboxylic acid.

CH HCN I OH (NHmC 03 C13 /0C2H5 C13 /OCzH OH CH 5 OH hydrolysis H NH CIONH: COQH (ilO-NH Reaction sequence V illustrates the preparation ofO-ethyl threonine from 2-ethoxypropion'aldehyde by reaction withammonium carbonate and hydrogen or alkali cyanide to form thecorresponding hyd antoin, followed by hydrolysis of the hydantoin to theamino acid.

In each of the foregoing synthesis, the O-ethyl threonine or derivativethereof is recovered as the racemic mixture. As noted above, the racemicmixture can be used in combatting PPLO organisms. However, it isdesirable to resolve the mixture to obtain substantially pure L-isomer,since that isomer possesses the highest degree of 'anti- PPLO activity.Resolution of the racemic mixture is most readily carried out by formingthe salt of the N-acyl O- ethyl threonine and an optically active base.The resulting diastereoisomers can be separated by fractionalcrystallization and the salt cleaved to yield the substantially pureL-isomer. The remaining D-isomer can be recovered and Esed, for example,as a resolving agent for optically active ases.

The N-acyl substituent can be any acyl moiety which can be s q y eavedand whi h does not interfere with the resolution, e.g. by altering thesolubility of the compound in an undesirable manner. Examples ofsuitable N-acyl substituted compounds are those of the formula NHR4CHgCIED-CHC OOH OCgHs wherein R is 'acyl, typically a lower alkanoyl,monocarbocyclic aroyl or aralkanoyl group such as formyl, acetyl,propionyl, butyryl, valeryl, caproyl, caprylyl, capryl, benzoyl, toluyl,phenylacetyl, phenylpropionyl and inertly substituted derivativesthereof. Examples of optically active bases which are useful inpreparing the diastereoisomer salts are dand l-a-phenylethylamine,cinchonine, quinine, quinidine, strychnine, brucine, morphine, 1-menthyl'amine, d-Z-amino-l-hydroxyhydrindine, L-arginine, and the like.

The N-acyl compounds are readily prepared from O- ethyl threonine byreaction with a reactive form of the desired acid, such as anhydride oracyl halide. The diastereoisomer salts are typically prepared byreaction of the free acid with an optically active amine, followed byfractional crystallization to separate the desired L-isomer salt. TheN-acyl compounds described above 'are useful in the preparation of thediastereoisomer salts which, in turn, are used to produce L-O-ethylthreonine and its anti-PPLO active derivatives.

The compounds having anti-PPLO activity can be administered tolivestock, especially fowl or poultry, in a number of ways. When usedprophylactically, the compounds are preferably administered in theanimals feed. Suitably, the compound will be given in a dosage of about0.005 to 0.15 gram per day per kilogram of body weight. The recommendeddoses are conveniently given by feeding the animal a feed compositioncomprising at least one element of the animals diet and about .005 to0.1% by weight of the compound. The lower amounts are generally suitablefor routine prophylactic use and the higher levels are more commonlyemployed in treating an established outbreak of the infection.Therapeutic doses are generally given in the animals water, bysubcutaneous injection, in a bolus or by drench gun.

The following examples represent illustrative methods for preparingrepresentative compounds of the invention.

EXAMPLE 1 DL-2-amino-3-ethoxybutyric acid To a mixture of 213 gm. ofmercuric acetate in one liter of anhydrous ethanol there is added 57.4gm. of crotonic acid and the mixture is heated until all of the solidsdissolve. The solution is then allowed to cool with stirring as theadduct precipitates. After 48 hours, the precipitate of2-acetoxy-mercuri-3-ethoxybutyric acid is filtered otf, washed withethanol and dried to give 177 gm. of product, melting point 103-105 C.

The adduct is dissolved in 600 ml. of water containing 101 gm. ofpotassium bromide and cooled to C. in an ice bath. A solution of 90 gm.of bromine and 101 gm. of potassium bromide in 168 ml. of water is addedslowly with rapid stirring while the surface of the solution isilluminated with a mecury vapor lamp. At the end of the addition, excessbromine is destroyed with a small amount of sodium bisulfite. Thesolution is acidified with 113 ml. of 48% hydrobromic acid and extractedwith four 400 ml. portions of ether. The combined ether extracts arewashed with 100 ml. of cold water, dried over sodium sulfate, andevaporated to dryness to give 103 gm. of 2- bromo-3-ethoxybutyric acid.

The 2-bromo compound is dissolved in 400 ml. of concentrated ammoniumhydroxide and heated in a bombtube for six hours at 100 C. Aftercooling, the excess ammonium hydroxide is evaporated and the residueplaced on a column containing one pound of Dowex 50 resin on thehydrogen cycle. The column is washed with water 6 until the effluent isneutral and is then eluted with 1 N ammonium hydroxide. The eluantfractions are recovered and tested with Ninhydrin for the present ofamino acid. The fractions giving a positive test are combined andevaporated to give a residue of DL-threo and erythro-Z-amino-3-ethoxybutyric acids.

EXAMPLE 2 DL-N-formyl-O-ethyl threonine 9 gm. of the crude mixtureobtained from Example 1 is dissolved in 100 ml. of formic acid and ml.of acetic anhydride is added. During the addition, the temperature risesto 65 C. The mixture is allowed to stand for one hour, and then isdiluted with ml. of Water and evaporated to dryness in vacuo. Water isadded and the mixture is taken to dryness several times to remove excessacid. Finally, a concentrated aqueous solution is cooled in an ice bathto crystallize. The threo-N-formyl-2-amino-3- ethoxybutyric acid(N-formyl-O-ethyl threonine) crystallizes out and is removed byfiltration and recrystallized from acetone to give a melting point of159-460 C.

EXAMPLE 3 DL-O-ethyl threonine A solution of 1.5 gm. of the product ofExample 2 in 20 ml. of 0.5 N hydrochloric acid is heated under refluxfor 1.5 hours, then placed on a column containing 50 gm. of Dowex 50resin on the hydrogen cycle. The column is washed with 500 ml. of waterand eluted with 1 N ammonium hydroxide. Fractions of the eluant giving apositive amino acid test with Ninhydrin are combined and evaporated todryness. The residue is stirred with ethanol and filtered to give 0.9gm. of DL-O-ethyl threonine, melting point 214-216 C.

EXAMPLE 4 L-N-formyl-O-ethyl threonine A solution is prepared from 4.5gm. of the product of Example 2, 3.1 gm. of d-wphenylethylamine, and 5ml. of hot methanol and 100 ml. of acetone is added thereto. Theresulting mixture is cooled to -10 C. and crystallization is induced byscratching the vessel. After two hours, the precipitate is filtered offand washed with acetone to give 1.6 gm. of the d-a-phenylethylamine saltof L(+)- N-formyl-O-ethyl threonine in the form of stout prisms, meltingpoint 135136 C. a |5.8 water (c. 1.0).

The filtrate is concentrated to one-third of its original volume,diluted with 50 ml. of acetone and again cooled to -10 C. to yield anadditional 1.2 gm. of the above salt, melting point 134135 C. Thecombined materials are dissolved in 10 ml. of 1 N sodium hydroxide andthe released d-a-phenylethylamine extracted with three 25 ml. portionsof chloroform. The aqueous layer is acidified with 10 ml. of 1 Nhydrochloric acid and cooled to yield crystals of L(-+)-N-formyl-O-ethylthreonine which are filtered off and washed with a small amount of icewater. The product has a melting point of 'l65-166 C. and u =14.3 water(c. 1.0).

It is sometimes found that the phenylethyl amine salt crystallizes in adiffered crystalline form, fine needle melting at -129 C., and this formis handled in the same manner as described above.

EXAMPLE 5 L-O-ethyl threonine The product of Example 4 is hydrolyzedwith dilute hydrochloric acid according to the procedure of Example 3 togive L-(-)-O-ethyl threonine, melting point 218- 219 C., u =49.2 water(c. 1.0). Either the pure L- isomer of this example or the racemicmixture of Example 3 can be converted, by known techniques, to the N-substituted and carboxyl-substituted derivatives previously described.

EXAMPLE 6 Methyl DL-O-ethyl threnonine Eighty mmoles (5.8 ml.) ofthionyl chloride is added dropwise with stirring to 35 ml. of methanolcooled to 5 C. in a salt-ice bath. To this solution is added 75 mmoles(11 gm.) of DL-O-ethyl threonine in portions, while the temperature ismaintained between and -6 C. The solution is allowed to come to roomtemperature then heated at 40 C. for two hours. The methanol isdistilled otf under reduced pressure and the residual clear, thickliquid dried for one hour in vacuo over a steam bath. The product isdissolved in ml. of water and the solution covered with 200 ml. ofether. Concentrated ammonium hydroxide is added until the pH of theaqueous layer reaches 8. The layers are separated and the organic layeris washed three times with 5 ml. of water, then dried over sodiumsulfate. Removal of the ether leaves a crude, pale yellow liquid whichis distilled to give methyl DL-O-ethyl threonine.

In a similar manner, the L-isomer of Example 5 can be substituted forthe racemic mixture to give methyl L-O- ethyl threonine.

EXAMPLE 7 DL-threo-2-amino-3-ethoxybutyramide A suspension of 60 mmoles(8.9 gm.) of DL-O-ethyl threonine in 375 ml. of dry dioxane is stirredover a water bath maintained at 40 C. Phosgene is introduced under thesurface of the liquid for 4-5 hours, the amino acid dissolvingcompletely within one hour. The reaction mixture is then sparged withnitrogen for twelve hours and the dioxane is stripped off in a rotaryevaporator at 40 C. The remaining colorless crystallineN-carboxyanhydride of O-ethyl threonine is washed with ether and driedon the filter.

A solution of 31 mmoles of the N-carboxyanhydride dissolved in 50 ml. ofdry tetrahydrofuran is added dropwise to 150 ml. of tetrahydrofuransaturated with ammonia over an ice bath, whereupon a white solid formsimmediately. Additional ammonia is bubbled in until no further reactionis observed. The solid is filtered off and the filtrate stripped oftetrahydrofuran to give a yellow liquid. The filtered solid is leachedwith hot ethyl acetate, filtered and the filtrate stripped of ethylacetate to give additional yellow liquid. The combined products aredistilled under vacuum to give DL-threo-2-amino-3-ethoxybutyramide.

In a similar manner, replacement of the DL-O-ethyl threonine by theL-isomer of Example 5 gives the L- isomer of the amide.

EXAMPLE 8 DL-threo-2-amino-3-ethoxybutyric acid hydrazide A mixture of0.04 mole of ester of DL-O-ethyl threonine and ml. of ethanol is heatedunder reflux with 2.0 ml. of hydrazine hydrate for three hours and isallowed to stand overnight. The solvent is stripped under vacuum to givea colorless oil which crystallizes on standing to give hygroscopiccrystals of DL-threo-2-amino-3- ethoxybutyric acid hydrazide.

EXAMPLE 9 DL-threo-2-amino-3-ethoxy-N-hydroxy-butyramide Separatesolutions of 96 mmoles of hydroxylamine hydrochloride in ml. of methanoland 144 mmoles of potassium hydroxide in 20 m1. of methanol are preparedat the boil. Both are cooled slightly and the hydroxide solution isadded, with stirring, to the hydroxylamine solution. The mixture is thencooled in an ice bath to complete precipitation of the potassiumchloride. A quantity of 48 mmoles of the methyl ester of DL-O-ethylthreonine is added with stirring and the mixture filtered immediately.The solid is washed with methanol and the combined filtrates are treatedwith an additional 48 mmoles of hydroxylamine hydrochloride toneutralize excess potassium hydroxide, and the solution is againfiltered. The filtrate deposits crystals on standing overnight. Thecrystals are collected and sublimed to give DL-threo-2-amino-3-ethoxy-N-hydroxybutyramide.

The unique anti-PPLO activity of the compounds of this invention isdemonstrated by the following.

EXAMPLE 10 A flock of chickens is artificially infected withpleuropneumonia-like organisms. Statistically significant groups ofchickens from this flock are then treated with each of the testcompounds, at a range of levels, by subcutaneous injection of thecompound in a sterile aqueous medium. The total dosage is given in twoseparate equal injections, one immediately after infection and the othersix hours later. All of the chickens from each group are tested forcontrol of the infection and the dosage level giving an average of 50%control in the group is calculated. The results obtained are as follows.

Dosage for 50% control 1N0 indication of control of infection at 250ing/kg. in any of the test animals.

From the results shown above, it can readily be seen that the compoundsof the invention are unique in their ability to combat PPLO infections,whereas their most closely related homologs and isomers are eithertotally devoid of activity or possess activity of such a low order thattheir use is not feasible.

EXAMPLE 1 1 Groups of eight day old White Leghorn chicks (10 birds pergroup) are infected with 1.0 ml. per bird of a culture of Mydoplasmagalliseptz'cum at dilutions of 10- 10- and 10- The infected chicks arefed a diet containing 0.02% by weight of L-O-ethylthreonine for a periodof ten days commencing 24 hours after infection and are sacrificed onthe eleventh day.

It is found that the 0.02% level of L-O-ethylthreonine is effective incontrolling the infection. Air sac exudate is completely eliminated inall chicks at all dilution levels. Similar results are observed when thecompound is administered in drinking water at a level of 0.04% byweight.

What is claimed is:

1. D-a-phenethylamine salt of L(+)-N-formyl-O-ethylthreonine.

References Cited UNITED STATES PATENTS 3,410,891 11/1968 Hughes260501.11

FOREIGN PATENTS 544,304 7/1957 Canada 260-534 OTHER REFERENCES Eliel:Stereochernistry of Carbon Compounds, Me- Graw-Hill Book Co., New York,pp. 49-51 (1962).

LEON ZITVER, Primary Examiner M. W. GLYNN, Assistant Examiner

